Antibody-dependent Cellular Cytotoxicity Reactions Research Articles

Key differences between anti-PD-1/PD-L1 inhibitors

Indications to immunotherapy in cancer treatment continue to expand, thus there are more and more questions about clinical aspects of using different checkpoint inhibitors. Despite similar mechanism of action between widely used antibodies to PD-1 (nivolumab, pembrolizumab, prolgolimab) and PD-L1 (durvalumab, avelumab, atezolizumab), inhibitors are different due to features of monoclonal antibodies structure they are based on. For instance, toxicity leading to discontinuation of treatment occurs more frequently with anti-PD-L1 drugs than PD-1 inhibitors. On the contrary, the average incidence of any grade IRAEs was higher in patients treated with anti-PD-1 drugs. The revealed differences in the toxicity of the analyzed groups of drugs could be associated with the type of action of the drug. The feature of the PD-L1 inhibitors is more frequent occurrence of antibody-dependent cellular cytotoxicity reactions. However, PD-1 inhibitors showed a statistically significant survival benefit, according to a meta-analysis comparing anti-PD-1 and anti-PD-L1 groups. Besides data on differences in the efficacy and toxicity profiles of these agents, in this article we also analyze different issues in the structure of drug molecules, in particular, the role of LALA mutation in anti-PD-1 inhibitors. Understanding the key distinctive points of check-point inhibitors (CPI) in the future may allow to solve the problem of rechallenge and reintroduction after management of severe IRAEs.

Downregulated ATP6V1B1 expression acidifies the intracellular environment of cancer cells leading to resistance to antibody-dependent cellular cytotoxicity.

Among several mechanisms for the resistance of human epidermal growth factor receptor 2-overexpressing (HER2 +) cancer cells to trastuzumab, little is known regarding the mechanism underlying the resistance to trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC). Cell death due to ADCC is caused by apoptosis of target cells induced by granzymes released from natural killer cells. Because optimal granzyme physiological activity occurs at neutral pH, we assumed that the pH of the intracellular environment influences the cytotoxic effects of granzymes. We established ADCC-resistant cells and compared them with wild-type cells in terms of the expression of intracellular pH-regulating genes. The expression of ATP6V1B1, which encodes a component of vacuolar ATPases, was downregulated in the ADCC-resistant cells. Thus, to functionally characterize ATP6V1B1, we used a CRISPR/Cas9 system to generate ATP6V1B1-knockout SKBR3 and JIMT-1 cells (both HER2 + human breast cancer cell line). The resulting cells exhibited significantly less ADCC than the control SKBR3 and JIMT-1 cells. The intracellular pH of the ATP6V1B1-knockout SKBR3 and JIMT-1 cells was significantly lower than control SKBR3 and JIMT-1cells. An analysis of granzyme dynamics during the ADCC reaction in cancer cells revealed that granzymes degraded intracellularly in the control SKBR3 and JIMT-1 cells and accumulated in ATP6V1B1-knockout cells, but were not cytotoxic. These findings suggest that decreased vacuolar ATPase activity alters the cytoplasmic pH of cancer cells to create an environment that is less suitable for granzyme bioactivity, which adversely affects the induction of apoptosis of cancer cells by NK cells.

Immunoregulatory role of B lymphocytes in alloresponse to kidney transplant.

B cells are a group of diverse phenotype and function subsets, which can both stimulate and inhibit the immune response to an allograft. They participate in the rejection process by influencing differentiation, proliferation and effector functions of T lymphocytes. B cells injure the graft via the ADCC (antibody-dependent cellular cytotoxicity) reaction and humoral rejection through plasmocyte production of donor-specific antibodies. A converse, suppressive mode of B cells can attribute to the development of tolerance and protect the graft from rejection. This function is provided by the regulatory B cells, which negatively control the immune response by producing suppressor cytokines (IL-10, IL-35, TGF-β), natural antibodies and through cellular interactions. In effect they inhibit the development of Th1 and Th17 effector cells, and induce differentiation of regulatory T cells. Operational immune tolerance in human kidney transplant recipients was associated with increased number of naïve and transitional B cells of regulatory function, and increased gene expression for differentiation of B cells. However, in chronic alloantibody transplant rejection the distorted distribution and function of regulatory B cells was found, which implies their pivotal role in graft tolerance. Currently, the immunosuppressive regimens unselectively inhibit the activity of T and B cells, by interfering with their effector and immunoregulatory functions. They do not fully control the chronic rejection reaction, which is the major cause of graft loss. Comprehension of the mechanisms of immunologic homeostasis dependent on B cells can help develop immunosuppressive protocols targeted at tolerance.

A monoclonal antibody against neem leaf glycoprotein recognizes carcinoembryonic antigen (CEA) and restricts CEA expressing tumor growth.

Carcinoembryonic antigen (CEA) is one of the promising tumor antigens mainly associated with carcinoma of the colon, lung, breast, etc. and received wide attention for cancer immunotherapy. Neem leaf glycoprotein (NLGP), an effective immunomodulator, is able to generate humoral and cellular immune responses in murine tumor models. We have generated a monoclonal antibody (mAb) against NLGP by fusing NLGP-immunized mice splenocytes with nonsecretory myeloma cells. A highly anti-NLGP mAb secreting clone (1C8; IgG2a in nature) has been identified and propagated in culture. 1C8 recognizes human CEA as good as NLGP by enzyme linked immunosorbent assay, Western blotting, and immunoprecipitation. 1C8 detects CEA on colon cancer tissues by immunochistochemistry. By flow cytometry, 1C8 specifically reacts with CEA(+) human (Colo-205, HCT-116, and HT-29) and mouse (CT-26) colon cancer cells, but it showed minimum reactivity with CEA(-) human (MCF7, SiHa, and SCC084) and mouse (B16MelF10) cancer cells. This anti-NLGP 1C8 mAb revealed significant antitumor activity and better survivability in vivo in animals bearing mouse (CT-26 in BALB/c) and human (Colo-205 in athymic nude) CEA(+) cancer cells. 1C8 has no direct influence on proliferation and migration of CEA(+) cells, however, NK cell-dependent strong antibody-dependent cellular cytotoxicity reaction toward CEA(+) cells and normalization of angiogenesis are chiefly associated with tumor growth restriction. Obtained results provided a new immunotherapeutic approach for the effective management of CEA(+) tumors.

Chapter Three - How Immunoglobulin G Antibodies Kill Target Cells: Revisiting an Old Paradigm

The capacity of immunoglobulin G (IgG) antibodies to eliminate virtually any target cell has resulted in the widespread introduction of cytotoxic antibodies into the clinic in settings of cancer therapy, autoimmunity, and transplantation, for example. More recently, it has become apparent that also the protection from viral infection via IgG antibodies may require cytotoxic effector functions, suggesting that antibody-dependent cellular cytotoxicity (ADCC) directed against malignant or virally infected cells is one of the most essential effector mechanisms triggered by IgG antibodies to protect the host. A detailed understanding of the underlying molecular and cellular pathways is critical, therefore, to make full use of this antibody effector function. Several studies over the last years have provided novel insights into the effector pathways and innate immune effector cells responsible for ADCC reactions. One of the most notable outcomes of many of these reports is that cells of the mononuclear phagocytic system rather than natural killer cells are critical for removal of IgG opsonized target cells in vivo.

Glycans in Sera of Amyotrophic Lateral Sclerosis Patients and Their Role in Killing Neuronal Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by degeneration of upper and lower motor neurons. To date, glycosylation patterns of glycoproteins in fluids of ALS patients have not been described. Moreover, the aberrant glycosylation related to the pathogenesis of other neurodegenerative diseases encouraged us to explore the glycome of ALS patient sera. We found high levels of sialylated glycans and low levels of core fucosylated glycans in serum-derived N-glycans of patients with ALS, compared to healthy volunteer sera. Based on these results, we analyzed the IgG Fc N297-glycans, as IgG are major serum glycoproteins affected by sialylation or core fucosylation and are found in the motor cortex of ALS patients. The analyses revealed a distinct glycan, A2BG2, in IgG derived from ALS patient sera (ALS-IgG). This glycan increases the affinity of IgG to CD16 on effector cells, consequently enhancing Antibody-Dependent Cellular Cytotoxicity (ADCC). Therefore, we explore whether the Fc-N297-glycans of IgG may be involved in ALS disease. Immunostaining of brain and spinal cord tissues revealed over-expression of CD16 and co-localization of intact ALS-IgG with CD16 and in brain with activated microglia of G93A-SOD1 mice. Intact ALS-IgG enhanced effector cell activation and ADCC reaction in comparison to sugar-depleted or control IgG. ALS-IgG were localized in the synapse between brain microglia and neurons of G93A-SOD1 mice, manifesting a promising in vivo ADCC reaction. Therefore, glycans of ALS-IgG may serve as a biomarker for the disease and may be involved in neuronal damage.

Effective Elimination of Acute Myeloid Leukemic Cells by Recombinant Bispecific Antibody Derivatives Directed Against CD33 and CD16

Single-chain Fv triplebodies (sctb), consisting of a single polypeptide chain with 3 single-chain antibody variable fragments connected in tandem, were generated as antileukemic agents. A CD19-specific sctb of this format has previously been shown to be superior to a bispecific single-chain Fv antibody fragment (bsscFv) for the elimination of leukemic B-lineage cells, but corresponding targeted agents for the treatment of acute myeloid leukemia are still lacking. For this purpose, both a bsscFv and a sctb specific for CD33 and the trigger molecule CD16 (FcgammaRIII) were produced. The sctb displayed 3.5-fold greater avidity for CD33 than the bsscFv 33xds16, whereas both had close to equal affinity for CD16. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, both the bsscFv 33xds16 and the sctb induced lysis of tumor cells with half maximum effective concentrations (EC50) in the low picomolar range. It is interesting to note that the sctb promoted equal lysis of human leukemia-derived cell lines at 10 to 200-fold lower concentrations than the bsscFv. Both molecules mediated ADCC of primary patient cells. In conclusion, both the bsscFv 33xds16 and the sctb 33xds16x33 eliminated acute myeloid leukemia cells in ADCC reactions, but the novel sctb format showed significantly greater specific activity.

Multiple molecular mechanisms underlying trastuzumab and lapatinib resistance in JIMT-1 breast cancer cells

Trastuzumab plays an important role in breast cancer therapy. However, a significant fraction of patients do not respond to therapy or they tend to develop resistance shortly after beginning therapy. Although some resistance mechanisms have been described, it is unclear whether these mechanisms can coexist. In this study, we analyzed the resistance mechanisms in the breast cancer cell line JIMT-1, a model of intrinsic trastuzumab resistance. We compared the JIMT-1 cell line with a panel of eight HER-2 positive breast cancer cell lines. All cell lines were characterized for the phosphatidylinositol 3-kinase ( PIK3CA) mutation status, expression levels of the phosphatase and tensin homolog on chromosome 10 (PTEN) and neuregulin-1 (NRG1) mRNA, HER-2 gene copy number, and protein expression. The results were correlated to the sensitivity to trastuzumab and lapatinib as well as the potency of trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) evoked by trastuzumab. JIMT-1 cells showed several co-existing drug resistance mechanisms, including an activating mutation of the PIK3CA gene, low expression of PTEN, high expression of NRG1, and relatively low expression of HER-2 receptor protein (despite gene amplification). All these features were present at variable levels in other cell lines, whereas JIMT-1 was unique in displaying all these factors at the same time. Unexpectedly, ADCC reaction by normal lymphocytes was equally strong in all HER-2 positive cell lines, without any correlation to molecular markers or direct sensitivity to the drugs. Resistance to trastuzumab and lapatinib is probably caused by several co-existing molecular mechanisms. Direct sensitivity to trastuzumab and lapatinib was not correlated with ADCC.

A Novel CD19-directed Recombinant Bispecific Antibody Derivative With Enhanced Immune Effector Functions for Human Leukemic Cells

A novel bispecific antibody-derived recombinant protein targeting leukemias and lymphomas was designed, a single-chain Fv triple body (sctb) consisting of 1 polypeptide chain with 3 scFvs connected in tandem. The distal scFvs were specific for the tumor antigen CD19, and the central scFv for the trigger molecule CD16 (FcgammaRIII) on natural killer (NK) cells and macrophages. We had previously built a disulphide stabilized (ds) bsscFv [19 x 16] with monovalent binding for CD19 from ds components. The sctb ds[19 x 16 x 19] also used ds components and displayed 3-fold greater avidity for CD19 than the bsscFv (KD = 13 vs. 42 nM), whereas both had equal affinity for CD16 (KD = 58 nM). Plasma half-lives in mice were 4 and 2 hours for the sctb and the bsscFv, respectively. In antibody-dependent cellular cytotoxicity reactions with human mononuclear cells as effectors, the sctb promoted equal lysis of leukemic cell lines and primary cells from leukemia and lymphoma patients at 10-fold to 40-fold lower concentrations than the bsscFv. This new format may also be applicable to a variety of other tumor antigens and effector molecules. With half-maximum effective concentrations (EC50) in the low picomolar range, the sctb ds[19 x 16 x 19] is an attractive candidate for further preclinical evaluation.

Trastuzumab causes antibody-dependent cellular cytotoxicity–mediated growth inhibition of submacroscopic JIMT-1 breast cancer xenografts despite intrinsic drug resistance

Trastuzumab is a recombinant antibody drug that is widely used for the treatment of breast cancer. Despite encouraging clinical results, some cancers are primarily resistant to trastuzumab, and a majority of those initially responding become resistant during prolonged treatment. The mechanisms of trastuzumab resistance have not been fully understood. We examined the role of antibody-dependent cellular cytotoxicity (ADCC) using JIMT-1 cells that are ErbB2 positive but intrinsically resistant to trastuzumab in vitro. Unexpectedly, in experiments mimicking adjuvant therapy of submacroscopic disease in vivo (JIMT-1 cells inoculated s.c. in severe combined immunodeficiency mice), trastuzumab was able to inhibit the outgrowth of macroscopically detectable xenograft tumors for up to 5-7 weeks. The effect is likely to be mediated via ADCC because trastuzumab-F(ab')(2) was ineffective in this model. Moreover, in vitro ADCC reaction of human leukocytes was equally strong against breast cancer cells intrinsically sensitive (SKBR-3) or resistant (JIMT-1) to trastuzumab or even against a subline of JIMT-1 that was established from xenograft tumors growing despite trastuzumab treatment. These results suggest that ADCC may be the predominant mechanism of trastuzumab action on submacroscopic tumor spread. Thus, measuring the ADCC activity of patient's leukocytes against the tumor cells may be a relevant predictor of clinical trastuzumab responsiveness in vivo.

Granulocyte Macrophage Colony–Stimulating Factor Expression in Human Herpetic Stromal Keratitis: Implications for the Role of Neutrophils in HSK

Granulocyte macrophage colony-stimulating factor (GM-CSF) is thought to play a key role in chronic inflammatory diseases by governing the survival and function of infiltrating neutrophils. The objective of this study was to determine the putative role of GM-CSF in the pathogenesis of human herpetic stromal keratitis (HSK). Primary human corneal fibroblast (HCF) cultures and a telomerase-immortalized human corneal epithelial (HCE) cell line representative of native HCE were stimulated with the known HSK-inducing cytokines interferon (IFN)-gamma, interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha. Alternatively, the T-cell cytokine IL-17 was added solely or simultaneously. Human neutrophils were incubated with conditioned medium (CM) of the HCF and HCE stimulated with the aforementioned cytokines, or recombinant GM-CSF, and their viability or activation status was determined by flow cytometry. GM-CSF and IL-8 secretion levels in the CM were determined by ELISA. The antibody-dependent cellular cytotoxicity (ADCC) of neutrophils toward herpes simplex virus (HSV)-infected HCFs was determined by flow cytometry. The expression of GM-CSF was determined in HSK and control corneal buttons by real-time RT-PCR and immunohistology. Compared with IFN-gamma, CM of either cell type stimulated with IL-1beta, or in the case of HCE cells, stimulated with TNF-alpha or IL-17, delayed neutrophil apoptosis significantly. Only in HCFs did IL-17 exhibit a synergistic effect with TNF-alpha. The antiapoptotic activity was attributable in part to the GM-CSF secreted by the activated HCFs and HCE cells. GM-CSF stimulation of neutrophils induced their activation and the secretion of IL-8. GM-CSF did not increase significantly the ADCC reaction of neutrophils toward HSV-infected HCFs. Finally, GM-CSF was expressed in corneas of the patients with HSK but not in control subjects. The data suggest that GM-CSF, expressed by cornea-resident cells such as HCFs and HCE cells, may play a role in the immunopathogenesis of HSK by prolonging the survival and modulating the effector function of corneal infiltrating neutrophils.

Apoptosis of human naive NK cells mediated by a rat IgG2b anti CD2 mAb through a fractricidal ADCC reaction

LO-CD2a/BTI-322, a rat anti human CD2 mAb, shows in vitro and in vivo immunosuppressive properties and induces T-cell depletion resulting partially from an antibody dependent cellular cytotoxicity (ADCC) mediated by NK cells. The aim of this paper is to study the in vitro effect of LO-CD2a/BTI-322 on NK cells, the majority of them also expressing the CD2 molecule. The addition of the mAb to purified naive NK cells induces apoptosis of CD2 + cells. The apoptosis is rapid, Fas ligand independent and completely inhibited by the calcium chelator EGTA, suggesting a fractricidal ADCC reaction and implying that NK cells are not resistant to lysis when used as target cells. At the end of the reaction, the CD2 − remaining cells are still capable of natural cytotoxicity against K562 cells, but at a lower rate than untreated cells.

FcγRII (CD32) Is Linked to Apoptotic Pathways in Murine Granulocyte Precursors and Mature Eosinophils

AbstractMurine granulocytes and precursors express low-affinity IgG Fc receptors (FcγR). We investigated the effects of FcγR ligation on the development of eosinophils in cultures of normal murine bone marrow. Eosinophilopoiesis was induced by culture of bone marrow cells in the presence of cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF], interleukin-3 [IL-3], and IL-5). Addition to the cultures of 2.4G2, a rat monoclonal antibody (mAb) that reacts with FcγRII (CD32) and FcγRIII (CD16), induced granulocyte apoptosis within 24 hours. Granulocytes in cultures that contained 2.4G2 showed chromatin condensation, binding of Annexin-V, and fas induction, and by electron microscopy, apoptosis was most commonly observed in cells of the eosinophil lineage. Since murine granulocytes can express both FcγRII (CD32) and FcγRIII (CD16), we investigated the effect of 2.4G2 on cultures of bone marrow obtained from FcγRIII (CD16) gene–disrupted mice and found that the apoptosis induced with 2.4G2 was CD16-independent. Studies with bone marrow cultures from B6MLR-lpr/lpr and C3H/HEJ-gld/gld mice established that the FcγRII (CD32)-triggered apoptosis was fas-fasL–dependent. When mature eosinophils isolated from hepatic granulomas of Schistosoma mansoni–infected mice were cultured in cytokines in the presence of 2.4G2, the eosinophils underwent apoptosis within 24 hours. These findings identify a previously unknown linkage between FcγR on eosinophils and fas-mediated apoptosis, a connection that could be relevant to mechanisms by which eosinophils mediate tissue injury and antibody-dependent cellular cytotoxicity reactions.

FcγRII (CD32) Is Linked to Apoptotic Pathways in Murine Granulocyte Precursors and Mature Eosinophils

Murine granulocytes and precursors express low-affinity IgG Fc receptors (FcγR). We investigated the effects of FcγR ligation on the development of eosinophils in cultures of normal murine bone marrow. Eosinophilopoiesis was induced by culture of bone marrow cells in the presence of cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF], interleukin-3 [IL-3], and IL-5). Addition to the cultures of 2.4G2, a rat monoclonal antibody (mAb) that reacts with FcγRII (CD32) and FcγRIII (CD16), induced granulocyte apoptosis within 24 hours. Granulocytes in cultures that contained 2.4G2 showed chromatin condensation, binding of Annexin-V, and fas induction, and by electron microscopy, apoptosis was most commonly observed in cells of the eosinophil lineage. Since murine granulocytes can express both FcγRII (CD32) and FcγRIII (CD16), we investigated the effect of 2.4G2 on cultures of bone marrow obtained from FcγRIII (CD16) gene–disrupted mice and found that the apoptosis induced with 2.4G2 was CD16-independent. Studies with bone marrow cultures from B6MLR-lpr/lpr and C3H/HEJ-gld/gld mice established that the FcγRII (CD32)-triggered apoptosis was fas-fasL–dependent. When mature eosinophils isolated from hepatic granulomas of Schistosoma mansoni–infected mice were cultured in cytokines in the presence of 2.4G2, the eosinophils underwent apoptosis within 24 hours. These findings identify a previously unknown linkage between FcγR on eosinophils and fas-mediated apoptosis, a connection that could be relevant to mechanisms by which eosinophils mediate tissue injury and antibody-dependent cellular cytotoxicity reactions.

Concomitant killing in vitro of both gp120-coated CD4+ peripheral T lymphocytes and natural killer cells in the antibody-dependent cellular cytotoxicity (ADCC) system.

NK cells play an important immunoregulatory role in first line defense mechanisms against infection. As disease progresses, HIV-1 infected patients show loss of NK cytotoxic function, down-modulation and/or loss of expression of both CD16 and CD56 surface Ags on NK cells and a gradual loss of both CD4+ T cells and NK cell numbers. A potential mechanism by which these manifestations may occur in vivo was investigated. We hypothesized that NK-mediated Ab-dependent cellular cytotoxicity (ADCC), using gp120-coated CD4+ peripheral T lymphocytes as targets and anti-HIV serum, will result in the concomitant killing of the CD4+ T lymphocyte targets and the NK lymphocytes. This hypothesis was examined in an in vitro model system. The findings demonstrate that gp120-coated peripheral T lymphocytes can serve as targets and are killed in ADCC. Further, the NK cells that recover from the ADCC reaction show a loss of cytotoxic function, acquire the CD16(dim/-) CD56(dim/-) phenotype and a significant fraction is killed by activation-induced cell death or apoptosis. These findings are reminiscent of the properties of circulating NK cells in HIV-infected patients. The implication of these findings in the pathogenesis of AIDS is discussed.

Epitopes of herpes simplex virus type 1 glycoprotein B that bind type-common neutralizing antibodies elicit type-specific antibody-dependent cellular cytotoxicity.

A panel of 45 well-characterized monoclonal antibodies (MAbs) reactive to glycoprotein B (gB) of herpes simplex virus (HSV) type 1 was tested by ELISA and in antiviral functional assays (that included virus neutralization, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent complement-mediated lysis), using type 1 or 2 virus strains. All MAbs were ELISA-reactive. Eleven of the MAbs mediated neutralization and 9 mediated ADCC. All of the ADCC epitopes were contained within the amino-terminal half of the extracellular portion of gB. The ADCC reactions were strictly type 1-specific, whereas 9 of 11 neutralizing MAbs exhibited type-common activity. There was some association between the ADCC and neutralization activities, since of 12 MAbs with functional activity, 8 were positive in both assays. These results suggest that differences in the presentation of gB, and perhaps HSV-1 gB versus HSV-2 gB, on free virus and virus-infected cells determine epitope availability.

Antibody-dependent cellular cytotoxicity detects type- and strain-specific antigens among human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus SIVmac isolates.

Human cell lines were infected with different strains of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) as well as with a simian immunodeficiency virus SIVmac isolate and used as targets in an antibody-dependent cellular cytotoxicity (ADCC) assay. Sera from HIV-1- or HIV-2-infected subjects provided the antibody, and lymphocytes from normal donors provided the effector cells. About 60% of HIV-1 antibody-positive sera mediated ADCC when tested against any given HIV-1 isolate-infected target cell (human T-cell lymphotropic virus type IIIB, B40, A2587), and about 75% of HIV-2 antibody-positive sera mediated ADCC when tested against target cells infected with HIV-2 isolates (lymphadenopathy-associated virus type 2 and SBL-6669) or simian immunodeficiency virus from macaques. Within each type, individual sera showed different reactivity patterns, and the probability that a serum was ADCC positive was higher when it was tested against several strains. When the ADCC reactivity of sera against different strains was compared, diversity as detected by ADCC appeared to be greater among HIV-1 strains than among HIV-2 strains. For HIV-1, 54 to 67% of the sera gave concordant ADCC reactions, whereas for HIV-2 and SIVmac, 91% of the sera gave concordant results. Almost no strain-specific differences were seen between SBL-6669 and lymphadenopathy-associated virus type 2. As we determined previously, HIV-1 and HIV-2 did not cross-react in ADCC. The results indicated that HIV-1 and HIV-2 antibody-positive sera mediate both strain- and type-specific ADCC. HIV-2 antibody-positive sera seem to mediate ADCC with broader reactivity and to a higher frequency compared with HIV-1 antibody-positive sera.

Monoclonal antibodies against epitopes on ganglioside GD2 and its lactones. Markers for gliomas and neuroblastomas.

Monoclonal antibodies (mAbs) BW 625 and BW 704, of the IgG3 isotype, bound to immunochemically indistinguishable epitopes on ganglioside II3(Neu-Ac)2-GgOse3-Cer. Despite this fact the mAbs showed a differential binding pattern on human glioma cell lines i.e. immunohistochemical data indicate that the detected epitopes are not identical. Furthermore, either mAb is able to mediate the antibody-dependent cellular cytotoxicity reaction (ADCC) and the human-complement-dependent cytotoxicity reaction (CDC) with epitope-expressing tumor cells. All cryopreserved tissue specimens from gliomas and neuroblastomas were immunohistochemically stained, whereas the other small round cell tumors of childhood, as well as melanomas and small-cell lung carcinomas, were essentially negative. Positive staining of normal cryopreserved tissues was restricted to amyelinic axons, Hassal's bodies and some connective tissue fibers in thymus and the tegumentary epithelium of skin. The high selectivity of mAb BW 704 for gliomas and neuroblastomas, the lack of cross-reactivity with major tissues and the strong ADCC and CDC potential argue for the use of mAb BW 704 in immunotherapy of neuroblastomas and gliomas.

Agonistic Effects of Anti‐CD2 and Anti‐CD16 Antibodies on Human Natural Killer Killing

Two monoclonal antibodies (MoAb), 9-1 (anti-CD2) and 3G8 (anti-CD16), were previously shown to enhance the cytotoxic activity of human natural killer (NK) cells. The present study examined the effect of 9-1 and 3G8 with different effector and target cells to determine whether they activate NK cells through a common mechanism. Analysis of purified lymphocyte subpopulations demonstrated that the CD3+CD16+CD3- NK effector cell population is enhanced by both antibodies, while purified CD2+CD16-CD3+ T cells are not activated by either antibody. Although both antibodies enhance killing of K-562 and Daudi, killing of T-cell lines is enhanced by 9-1 and inhibited by 3G8. In contrast, killing of the promyelocytic cell line, U-937 is inhibited by 9-1 and enhanced by 3G8. On NK-susceptible cells the pattern of enhancement with 3G8, an IgG1 MoAb, is consistent with the pattern of target cell expression of an Fc receptor, FcR II, known to bind IgG1 antibodies. This suggests that 3G8 may cross-link effector and target cells through CD16 on the effectors and FcR II on these targets. This could activate NK killing by a mechanism similar to antibody-dependent cellular cytotoxicity reactions (ADCC) with the MoAb in the reverse orientation. The failure of 3G8 F(ab')2 fragments to enhance NK killing, further supports the reverse ADCC mechanism of enhancement by 3G8. The pattern of enhancement mediated by 9-1, an IgG3 MoAb, is not correlated with any target cell Fc-receptor known to bind IgG3 MoAb. The effect of 9-1 may result, instead, from its binding to the unique 9-1 epitope on the CD2 molecule involved in CD2-mediated T-cell activation, as previously described. Alternative mechanisms, including activation of NK killing by 9-1 mediated cross-linking of CD2 and CD16 on the effector cells, have also been discussed.

Role of oxygen-dependent mechanisms in monoclonal-antibody-induced lysis of normal T cells by phagocytes: II.—Murine phagocytes

In the first part of this study, we reported that antibody-dependent cellular cytotoxicity (ADCC) mediated by human polymorphonuclear (PMN) and mononuclear (MN) phagocytes against anti-T-cell monoclonal-antibody-(mAb)-coated T lymphocytes, is mainly dependent upon the generation of reactive oxygen intermediates (ROI) for PMN, whereas MN-mediated ADCC depends on both oxidative and non-oxidative cytolytic events. Using mouse effector cells from various organs and at various maturation or activation states, the present report shows that in this ADCC model against mAb-coated normal T cells, resident spleen (SPC) and peritoneal exudate (PEC) cells selectively bound and developed oxidative responses to these mAb-coated normal target cells but remained ineffective in ADCC. Similar data were obtained with inflammatory recruited (thioglycollate-elicited or Biogel granuloma-induced) macrophages, whereas immunologically activated macrophages (from BCG-treated mice) mediated both strong oxidative responses and potent ADCC reactions. In contrast, and as in the human system, both resident (from bone marrow) and inflammatory PMN phagocytes exerted significant lysis of these mAb-sensitized normal lymphoid cells. These findings, which are similar to those reported in ADCC against tumour target cells, strongly suggest that the nature (normal or tumoral) of the target cell does not influence the lytic